Zizaane-Type Sesquiterpenoids and Their Rearranged Derivatives from Agarwood of an Aquilaria Plant

Nine new sesquiterpenoids (1–9) were isolated from ethyl ether extract of agarwood originated from Aquilaria sp., including three novel sesquiterpenoids (1–3) derived from zizaane, together with six zizaane-type sesquiterpenoids (4–9). All structures were unambiguously elucidated based on 1D and 2D NMR spectra as well as by HRESIMS data. The absolute configuration of sesquiterpenoids was determined by comparison of the experimental and computed ECD spectra. In vitro anti-inflammatory assessment showed that compound 9 exhibited inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC50 value of 62.22 ± 1.27 μM.

Compound 6 was isolated as a white amorphous powder. The molecular formula of C 15 H 24 O 2 was deduced from its HRESIMS, differing from 5 by the loss of one oxygen and the addition of two extra protons. Comparison of its 1 H and 13 C NMR data (Tables 3 and 4) with those of compound 5 showed an additional methylene group (δ H 2.31, 2.16 and δ C 28.2, CH 2 -4) and the disappearance of keto group in 5, which was confirmed by COSY correlations between H 2 -12 (δ H 3.63 and 3.45)/H-2 (δ H 2.12)/H 2 -3 (δ H 1.75 and 1.30)/H 2 -4. The remaining structure of 6 was shown to be identical to that of 5 by detailed analysis of the 2D NMR spectra of 6.
Compound 8 shared the same planar structure as that of 7 by detailed analysis of its HRESIMS and 2D NMR spectrum. The coupling constant of H-4 (δ H 4.24, t, J = 6.4 Hz) was comparable to that of (4R)-albaflavenol (δ H 4.61, t, J = 7.8 Hz), suggesting the cis configuration between H-2 (δ H 2.02) and H-4 [6]. Meanwhile, the key NOE correlation between H-2 and H-4 in the ROESY spectrum suggested that 7 and 8 are 4-epimers.
To elucidate the absolute configuration of 1, 3 and 4, the solution TDDFT-ECD method was applied [7,8]. The initial Merck Molecular Force Field (MMFF) conformers were reoptimized at the B3LYP/6-31+G(d,p) in vacuo and the CAM-B3LYP/TZVP PCM/MeOH levels, separately, and ECD spectra were computed with four different functionals for the low-energy conformers. In the case of proposed (1S,2S,4S,6R,8R,11R)-3, a single conformer (Supplementary Materials) was found for which all combinations of levels gave moderate to good agreement with the experimental spectrum based on which the absolute configuration could be unambiguously elucidated as (1S,2S,4S,6R,8R,11R) ( Figure 4). It is interesting to note that the B3LYP and PBE0 functionals performed better for the minor transitions in the low-wavelength region then the CAM-B3LYP and BH&HLYP ones.
To elucidate the absolute configuration of 1, 3 and 4, the solution TDD method was applied [7,8]. The initial Merck Molecular Force Field (MMFF) con were reoptimized at the B3LYP/6-31+G(d,p) in vacuo and the CAM-B3LY PCM/MeOH levels, separately, and ECD spectra were computed with four d functionals for the low-energy conformers. In the case of proposed (1S,2S,4S,6R,8 3, a single conformer (Supplementary Materials) was found for which all combina levels gave moderate to good agreement with the experimental spectrum based o the absolute configuration could be unambiguously elucidated as (1S,2S,4S,6R (Figure 4). It is interesting to note that the B3LYP and PBE0 functionals performe for the minor transitions in the low-wavelength region then the CAM-B3L BH&HLYP ones. In the case of 1, the B3LYP and PBE0 functionals which performed better fo a mismatch in all applied combinations for the proposed (5R,6S,8R,11R) enantiom the BH&HLYP and CAM-B3LYP functionals reproduced all experimental transiti ( Figure 5). Analysis of the distinct conformers indicated that a different Boltzmann can reproduce the experimental spectrum also in the case of the B3LYP an functionals which can derive from a moderate error in estimating the relative ene the applied levels of theories [9,10]. Therefore the initial 3 MMFF conformers w reoptimized at the SOGGA11-X/TZVP [11]. SMD/MeOH level. The SOG functional was found one of the best in a recent DFT benchmark study [12]. Ind computing ECD for the SOGGA11-X conformers (Supplementary Materials), applied TDDFT functionals gave good agreement with the experimental spectrum In the case of 1, the B3LYP and PBE0 functionals which performed better for 3 gave a mismatch in all applied combinations for the proposed (5R,6S,8R,11R) enantiomer while the BH&HLYP and CAM-B3LYP functionals reproduced all experimental transitions well ( Figure 5). Analysis of the distinct conformers indicated that a different Boltzmann-weight can reproduce the experimental spectrum also in the case of the B3LYP and PBE0 functionals which can derive from a moderate error in estimating the relative energies by the applied levels of theories [9,10]. Therefore the initial 3 MMFF conformers were also reoptimized at the SOGGA11-X/TZVP [11]. SMD/MeOH level. The SOGGA11-X functional was found one of the best in a recent DFT benchmark study [12]. Indeed, by computing ECD for the SOGGA11-X conformers (Supplementary Materials), all four applied TDDFT functionals gave good agreement with the experimental spectrum in line with the biosynthetic considerations allowing elucidation of the absolute configuration as (5R,6S,8R,11R)-1. The relative intensities of the transitions were reproduced better by the BH&HLYP and CAM-B3LYP functionals than by the other two. with the biosynthetic considerations allowing elucidation of the absolute configur (5R,6S,8R,11R)-1. The relative intensities of the transitions were reproduced bette BH&HLYP and CAM-B3LYP functionals than by the other two. For proposed (1R,2S,8S)-4, both the gas-phase and the solvent model calc gave acceptable to good agreements with the experimental ECD spectrum (Fi Similarly to 1, ECD spectra computed with the B3LYP and PBE0 functional rep all transitions well, while the CAM-B3LYP and especially the BH&HLYP functio problems with the reproduction of the negative ECD transition at 260 nm. Repro of the other two transitions and nice agreement with the B3LYP and PBE0 fun however, allowed elucidation of the absolute configuration as (1R,2S,8S), which i line with the biosynthetic considerations. To improve the agreement, DFT optim were also performed at the SOGGA11-X/TZVP SMD/MeOH level but very simila were found to those of the gas-phase and PCM calculations. The ECD calculations compounds emphasize further that it is always advisable to apply more than o functional both for the DFT optimization and the TDDFT calculation steps [7 similar ECD spectra of 4 and 5 (the latter was noisy in the low-wavelength regio elucidation of the AC of 5 as (1R,2S,8R,11R). The absolute stereochemistry of the compounds 6-9 was determined based on biosynthetic considerations. For proposed (1R,2S,8S)-4, both the gas-phase and the solvent model calculations gave acceptable to good agreements with the experimental ECD spectrum (Figure 6). Similarly to 1, ECD spectra computed with the B3LYP and PBE0 functional reproduced all transitions well, while the CAM-B3LYP and especially the BH&HLYP functionals had problems with the reproduction of the negative ECD transition at 260 nm. Reproduction of the other two transitions and nice agreement with the B3LYP and PBE0 functionals, however, allowed elucidation of the absolute configuration as (1R,2S,8S), which is also in line with the biosynthetic considerations. To improve the agreement, DFT optimizations were also performed at the SOGGA11-X/TZVP SMD/MeOH level but very similar results were found to those of the gas-phase and PCM calculations. The ECD calculations of these compounds emphasize further that it is always advisable to apply more than one DFT functional both for the DFT optimization and the TDDFT calculation steps [7][8][9]. The similar ECD spectra of 4 and 5 (the latter was noisy in the low-wavelength region) allow elucidation of the AC of 5 as (1R,2S,8R,11R). The absolute stereochemistry of the further compounds 6-9 was determined based on biosynthetic considerations.
To our knowledge, this is the first time that so many ziaane-type sesquiterpenes were identified from agarwood, which may be the characteristic chemicals of this agarwood. A plausible biosynthetic pathway of sesquiterpenoids from agarwood is proposed to start from a liner precursor farnesyl pyrophosphate (FPP) modified after literature (Figure 7) [13,14]. The FPP undergoes ionization, cyclization, hydride shift, spirocyclization, cyclization and syn-1,2-methyl migration to generate the intermediate cation A. The formation of zizaanes 4-9 has been proposed to involve A, syn-deprotonation and oxidations of intermediated cation C. In addition, intermediated cation A undergoes rearrangement reactions a or b leading to cations B or C, which were catalyzed to form novel compounds 1 or 2, respectively. A biosynthetic pathway for 3 is proposed from 5 through 4,5-Bayer-Villiger oxidation, 4,5-hydrolysis and 5,6-enol-keto-tautomerization, 4,11-esterification and 4,12-condensation. To our knowledge, this is the first time that so many ziaane-type sesquiterp identified from agarwood, which may be the characteristic chemicals of this ag plausible biosynthetic pathway of sesquiterpenoids from agarwood is propos from a liner precursor farnesyl pyrophosphate (FPP) modified after literature [13,14]. The FPP undergoes ionization, cyclization, hydride shift, spiroc cyclization and syn-1,2-methyl migration to generate the intermediate catio formation of zizaanes 4-9 has been proposed to involve A, syn-deproton oxidations of intermediated cation C. In addition, intermediated cation A rearrangement reactions a or b leading to cations B or C, which were catalyze novel compounds 1 or 2, respectively. A biosynthetic pathway for 3 is propos through 4,5-Bayer-Villiger oxidation, 4,5-hydrolysis and 5,6-enol-keto-tautom 4,11-esterification and 4,12-condensation.  To our knowledge, this is the first time that so many ziaane-type sesquiterpenes were identified from agarwood, which may be the characteristic chemicals of this agarwood. A plausible biosynthetic pathway of sesquiterpenoids from agarwood is proposed to start from a liner precursor farnesyl pyrophosphate (FPP) modified after literature (Figure 7) [13,14]. The FPP undergoes ionization, cyclization, hydride shift, spirocyclization, cyclization and syn-1,2-methyl migration to generate the intermediate cation A. The formation of zizaanes 4-9 has been proposed to involve A, syn-deprotonation and oxidations of intermediated cation C. In addition, intermediated cation A undergoes rearrangement reactions a or b leading to cations B or C, which were catalyzed to form novel compounds 1 or 2, respectively. A biosynthetic pathway for 3 is proposed from 5 through 4,5-Bayer-Villiger oxidation, 4,5-hydrolysis and 5,6-enol-keto-tautomerization, 4,11-esterification and 4,12-condensation.  All isolated sesquiterpenes (1-9) were tested for α-glucosidase inhibition and antiinflammatory activities in vitro. Acarbose was used as a positive control for α-glucosidase inhibition with an IC 50 value of 743.4 ± 3.3 µM. Quercetin and indomethacin were used as positive controls for anti-inflammatory activity with IC 50 values of 8.22 ± 0.80 µM and 35.40 ± 1.77 µM, respectively. However, none of them exhibited α-glucosidase inhibition activity. Only compound 9 exhibited weak inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC 50 value of 62.22 ± 1.27 µM (Figure 8). The two hydroxy groups located at C-13 and C-15 effectively enhanced its anti-inflammatory activity. Although the anti-inflammatory activity of compound 9 is weaker than in the positive controls, this is the first report about the anti-inflammatory activity of ziaane-type sesquiterpenes. Further research is needed to confirm the activity and to uncover its exact mechanisms. In order to investigate whether the inhibitory activities of isolated sesquiterpenes were due to the decrease of cell numbers (cytotoxicity), their effects on cell viability also had been measured using the MTT method. None of them (up to 100 µM) showed cytotoxicity with LPS treatment.
35.40 ± 1.77 μM, respectively. However, none of them exhibited α-glucosidase inhibition activity. Only compound 9 exhibited weak inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC50 value of 62.22 ± 1.27 μM (Figure 8). The two hydroxy groups located at C-13 and C-15 effectively enhanced its anti-inflammatory activity. Although the anti-inflammatory activity of compound 9 is weaker than in the positive controls, this is the first report about the anti-inflammatory activity of ziaane-type sesquiterpenes. Further research is needed to confirm the activity and to uncover its exact mechanisms. In order to investigate whether the inhibitory activities of isolated sesquiterpenes were due to the decrease of cell numbers (cytotoxicity), their effects on cell viability also had been measured using the MTT method. None of them (up to 100 μM) showed cytotoxicity with LPS treatment.

Plant Material
The plant material was collected in NANA International Agarwood Market of Thailand, in August of 2014, and identified as agarwood originated from Aquilaria sp. by Dr. Jun Wang (Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences & Hainan engineering research center of agarwood). A voucher specimen (201408SLLK) has been deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.